Tissue specific expression of exogenous proteins in transgenic chickens

ABSTRACT

Transgenes encoding exogenous proteins are stably integrated into embryonic stem cells and are present in the somatic tissue of transgenic or chimeric birds. The transgenes encode exogenous proteins and are expressed in any of endodermal, ectodermal, mesodermal, or extra embryonic tissue. Tissue specificity is provided by selecting the content of the transgene accordingly. Transgenic birds whose genome is comprised of trangene derived exogenous DNA express exogenous proteins with tissue specificity, and specifically express exogenous proteins in the tubular gland cells of the oviduct to concentrate exogenous proteins in egg white.

FIELD OF THE INVENTION

This invention relates to the field of protein expression in transgenicand chimeric animals, and enabling technologies such as geneticengineering, transgenics, and the long-term culture of embryonic stemcells. Embryonic stem cells are engineered with specially designedgenetic constructs to introduce genetic modification into birds,including by the insertion of transgenes that yield tissue specificexpression of exogenous proteins. Transgenic birds of the inventionexpress the transgene-derived exogenous protein in the oviduct and theprotein is deposited in large quantities in the egg.

BACKGROUND OF THE INVENTION

Genetically engineered animals offer the potential for tremendousadvances in the production of valuable pharmaceutical products from thecells of such animals. However, the production of genetically modifiedanimals involves significant technical hurdles that have only beenovercome for a few species. The ability to incorporate geneticmodifications into the permanent DNA of a species requires severaldistinct technologies that must be developed for each geneticallyengineered species. One approach to alter the genetic and physicalcharacteristics of an animal is to use embryonic stem cells thatcontribute to the phenotype of an animal when injected into an embryonicform of the animal. Embryonic stem cells have the ability to contributeto the tissue of an animal born from the recipient embryo and tocontribute to the genome of a transgenic organism created by breedingchimeras.

Significant expenditure of time and resources has been committed to thestudy and development of embryonic stem cell lines, the manipulation ofthe genome of the cells, and cell culture techniques that permit suchengineered cells to be maintained in culture. Although many attemptshave been made, the ability to sustain the pluripotency of engineeredembryonic stem cells in culture has been achieved for only a fewspecies, notably mice. For other species, the promise of geneticengineering in transgenics for protein production has been frustrated bythe lack of sustainable long-term embryonic stem cell cultures.

If sustainable cultures of embryonic stem cells were readily availableand susceptible to genetic engineering while maintaining pluripotency, abroad application of new technologies would be available. Becauseembryonic stem cells contribute to the permanent DNA of an animal, thephysiological characteristics of the animal from which an embryonic stemcell was derived can be transferred to a recipient embryo byincorporating these cells into the recipient animal in an embryonicstate. This offers two principal advantages: first, the phenotype of ananimal from which embryonic stem cells are derived can be selectivelytransferred to a recipient embryo. Second, as noted above, when theembryonic stem cell cultures are particularly stable, the genome of thecells can be modified genetically to introduce genetic modificationsinto a recipient embryo in which the cells are introduced.

In certain cases, the embryonic stem cells can be engineered with atransgene that encodes an exogenous protein. The transgene is a geneticconstruct that contains DNA that acts as the blueprint for theproduction of a valuable protein and contains sufficient coding andregulatory elements to enable the expression of the protein in thetissue of the animal that is created from the insertion of the stemcells into a recipient embryo. In many cases, the expression of aprotein is particularly valuable because the protein can be collectedand isolated from the transgenic animal. However, the collection of avaluable protein from the tissues of an animal typically requires thatthe expression be limited to certain specific tissues that facilitatecollection of the expressed protein. For example, in cows, theexpression of a protein in the milk would enable the ready collection ofthe protein by simply collecting the milk of the cow and separating theexogenous protein. In chickens, the robust production of proteins in thewhite of the egg also provides an attractive vehicle for the expressionof exogenous proteins. If the expression of valuable proteins could beachieved in this manner, the animal could be used as a vehicle forproduction of proteins that is superior to other production methods.Thus, one particularly attractive field of research and attractive areafor commercial development is genetically engineered animals thatexpress selected exogenous proteins in specific tissue that facilitateisolation and collection of the protein. The ability to produceexogenous proteins in specifically selected cells of an animal is alsoparticularly valuable because the absence of tissue specificity simplyresults in the protein being expressed in all of the tissues of ananimal. Under such circumstances, it is unlikely that a meaningfulquantity of the protein could be separated from the animal, andfurthermore the ubiquitous expression of an exogenous protein is usuallyvery damaging to the overall health and well being of the animal.

If an embryonic stem cell culture is sufficiently stable to allow atransgene to become integrated into the genome of the embryonic stemcell, a transgene encoding tissue specific expression of a protein canbe passed to a new chimeric or transgenic organism by several differenttechniques depending on the specific construct used as the transgene.Whole genomes can be transferred by cell hybridization, intactchromosomes by microcells, subchromosomal segments by chromosomemediated gene transfer, and DNA fragments in the kilobase range by DNAmediated gene transfer (Klobutcher, L. A. and F. H. Ruddle, Annu. Rev.Biochem., 50: 533-554, 1981). Intact chromosomes may be transferred toan embryonic stem cell by microcell-mediated chromosome transfer MMCT)(Fournier, R. E. and F. H. Ruddle, Proc. Natl. Acad. Sci. U.S.A., 74:319-323, 1977). The specific design of the transgene also must considerthe content of the DNA sequences encoding the exogenous protein, thespecific tissue in which expression is targeted, the host organism inwhich expression occurs, and the nature of the protein to be expressed.Because proteins ordinarily expressed in vivo vary dramatically in theirsize, biochemical characteristics, and functionality, the transgenedesigned for tissue specific expression must satisfy several parametersto enable successful integration into the genome of an embryonic stemcell and to insure successful expression in the selected tissue of thehost organism.

As noted above, the performance of genetic modifications in embryonicstem cells to produce transgenic animals has been demonstrated in only avery few species. For mice, the separate use of homologous recombinationfollowed by chromosome transfer to embryonic stem (ES) cells for theproduction of chimeric and transgenic offspring is well known. Powerfultechniques of site-specific homologous recombination or gene targetinghave been developed (see Thomas, K. R. and M. R. Capecchi, Cell 51:503-512, 1987; review by Waldman, A. S., Crit. Rev. Oncol. Hematol. 12:49-64, 1992). Insertion of cloned DNA (Jakobovits, A., Curr. Biol. 4:761-763, 1994), and manipulation and selection of chromosome fragmentsby the Cre-loxP system techniques (see Smith, A. J. et al., Nat. Genet.9: 376-385, 1995; Ramirez-Solis, R. et al., Nature 378: 720-724, 1995;U.S. Pat. Nos. 4,959,317; 6,130,364; 6,091,001; 5,985,614) are availablefor the manipulation and transfer of genes into murine ES cells toproduce stable genetic chimeras. Many such techniques that have proveduseful in mammalian systems would be available to be applied tonon-mammalian embryonic stem cells if the necessary cell cultures wereavailable and if transgenes could be designed that yielded tissuespecific expression in specific tissues that facilitate isolation andcollection of the exogenous protein.

The transgenes that enable tissue specific expression are complex andthe genetic manipulations that are necessary to incorporate thetransgenes into an embryonic cell line require extensive manipulation ofthe embryonic stem cells and can threaten the pluripotency of the stemcells unless the culture conditions are optimized for transgenesis.Thus, embryonic stem cell lines suitable for use in transgenesis must beboth stable in culture and must maintain pluripotency when the ES cellis transfected with a genetic construct that is large and complex enoughto contain all of the elements necessary for protein expression.Moreover, the genetic construct must be expressed in the ES cell toallow selection of successfully transformed cells, and the ES cell mustmaintain potency and the transgene must remain viable during theinjection into recipient embryos and the formation of resulting animals.In the resulting animal, the transgene must be effectively expressed inspecific individual tissue types in which the transgene is designed tobe expressed, and, should not be expressed in other tissues such thatthe viability of the animal is compromised. For example, transgenesencoding DNA derived from the lymphoid elements of the immune systemmight be specifically targeted to be expressed in B lymphocytes of achimeric or transgenic animal. Specifically for the expression ofvaluable proteins, the transgene may be designed to express protein inthe oviduct such that the resulting protein is deposited in egg white.In an avian species, tissue-specific expression of exogenous proteins inthe cell types of the oviduct would yield a valuable biological systemfor protein production.

For the production of exogenous proteins, avian biological systems offermany advantages including efficient farm cultivation, rapid growth, andeconomical production. Globally, chickens and turkeys are a major sourceof protein in the human diet. Further, the avian egg offers an idealbiological design, both for massive synthesis of a few proteins and easeof isolation and collection of protein product. However, application ofthe full range of mammalian transgenic techniques to avian species hasbeen unsuccessful. Most notably, the transmission to a mature, livinganimal of a genetic modification encoding an exogenous proteinintroduced into an avian embryonic stem cell and expressed with tissuespecificity has not been demonstrated.

In many cases, the techniques necessary to introduce geneticmodifications into embryonic stem cells, the screening of modifiedembryonic stem cells to select specific cell modifications in which thegenetic constructs have been introduced, and the ability to manipulatethe ES cells for injection into embryos to produce transgenic chickens,requires at least several weeks for all of the steps to be performed.For the embryonic stem cells to be useful in transgenesis, thepluripotential state must be maintained for the entire time period upuntil injection into an embryo and the ES cell must be incorporated intoa recipient embryo to a degree necessary to express meaningfulquantities of the exogenous protein in the resulting animal.

SUMMARY OF INVENTION

The present invention includes transgenic and chimeric birds exhibitingnon-specific and tissue specific expression of exogenous proteins,transgene constructs that enable exogenous protein expression, isolatedcompositions of exogenous proteins, and related methods. The inventionuses long term avian ES cell cultures and special techniques to producechimeric and transgenic birds derived from prolonged embryonic stem cellcultures, wherein the genome of the ES cells have a stably integratedtransgene expressing an exogenous protein such that progeny of the EScells contain the transgene. In some embodiments, these geneticconstructs modify the DNA of the ES cell to facilitate tissue specificexpression of an exogenous protein. When combined with a host avianembryo, by the procedures described below, those modified ES cellsproduce chimeric birds that incorporate the transgene into specific,selected somatic tissue of the resulting animals. These chimeric ortransgenic birds exhibit an ES-cell derived phenotype and express theforeign protein across all tissues or in a selected tissue. Preferably,a specific expression pattern focuses expression in a tissue or tissuetype to the substantial exclusion of other tissues and facilitatesconcentration and collection of the protein.

This invention also includes compositions comprising long-term culturesof chicken embryonic stem cells that have been genetically modified toincorporate large amounts of foreign DNA, and that contribute to thesomatic tissues and the germline of recipient embryos. The inventionalso includes transgenic chickens expressing exogenous protein in theoviduct tissue such that exogenous protein is concentrated in the eggwhite. In one preferred embodiment, the exogenous protein is amonoclonal antibody encoded by the transgene construct incorporated intothe genome of the embryonic stem cell and progeny. The monoclonalantibody sequence is contained within a transgene that is specificallyconstructed for expression in the oviduct and which contains appropriatepromoters and regulatory sequences to facilitate tissue specificexpression. In the embodiment of a transgenic or chimeric birdexpressing exogenous proteins, the invention includes compositionsspecific to the animal and the protein, such as egg white, albumencontaining exogenous proteins. For all of these embodiments, methods ofusing the constructs, animals, and exogenous proteins of the inventionare also included.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the characteristic morphology of chicken ES cells where thesmall cells grow in a single layer with little cytoplasm and a prominentnucleolus.

FIG. 2 shows in vitro properties of chicken ES cells, specifically thereaction with the antibodies SSEA-1 and EMA-1, and expression ofalkaline phosphatase.

FIG. 3 is a karyotype of chicken ES cells that have been in culture for189 days. The cells are diploid, carry 38 pairs of autosomal chromosomesand one pair of Z chromosomes.

FIG. 4 is two Barred Rock chicks and two chimeras formed by injectingBarred Rock ES cells into a White Leghorn recipient embryo. The chimerasand the Barred Rocks are indistinguishable indicating that thecontributions of the ES cells to the melanocyte lineage is extensive.

FIG. 5 are chimeras made by injecting Barred Rock ES cells into WhiteLeghorn recipients. The pair of chimeras in the left panel exhibit minorcontributions to the melanocyte lineage whereas the pair in the leftpanel show more extensive contributions.

FIG. 6 is a diagram of the pCX/GFP/Puro plasmid construct used fortransfection of ES cells.

FIG. 7 shows chicken ES cells that have been transfected with thepCX/GFP/Puro construct and grown in the presence of puromycin. The upperpanel is photographed to reveal fluorescence; the lower panel is thesame field viewed by phase contrast microscopy.

FIG. 8 is a FACS analysis of non-transfected chicken ES cells (upperpanel) and chicken ES cells that have been transfected with thepCX/GFP/Puro construct and grown in the presence of puromycin. Theanalysis shows that substantially all of the transfected cells areexpressing the transgene.

FIG. 9 is a Southern analysis of ES cells that have been transfectedwith the pCX/GFP/Puro construct. The difference in the location of theprobe in preparations of DNA digested with BamH1, EcoR1 and acombination of the two endonucelases indicates that the transgene inincorporated into the genome at different sites in the cell lines TB01and TB09.

FIGS. 10A-10D are a transgene construct providing tissue specificexpression in the tubular gland cells of the oviduct of a chicken andphysiological evidence confirming the tissue specific expression. FIG.10A is a diagram of a transgene designated Ov7.5MAbdns (upper panel);(B) A diagrammatic representation of a tissue-specific expressiontransgene containing DNA sequences derived from the host ovalbuminpromoter combined with coding DNA for a monoclonal antibody. FIG. 10B isa section of the magnum of a chimeric chicken containing thetissue-specific expression transgene showing the expression ofmonoclonal antibody selectively in tubular gland cells to the exclusionof other cell types. FIGS. 10C and 10D are RT-PCR analyses showingexpression of both the light and heavy chain of a monoclonal antibody inthe oviduct, to the exclusion of brain, gut, pancreas and muscle tissueof the chimeras.

FIG. 11 is the genomic PCR analysis of BAC-A transfected cES cells.

FIG. 12 is a Southern analysis of chimeras made with cES cellstransfected with CX/GFP/Puro.

FIG. 13 is a photograph of a chimeric chicken made with cES cellstransfected with CX/GFP/Puro under fluorescent light (left panel) andwhite light (right panel). Green fluorescence can be observed in theeyes and beak, confirming that the transfected ES cells contributed tothese tissues.

FIG. 14 is a photograph of a chimeric chicken made with cES cellstransfected with CX/GFP/Puro under fluorescent light. The greenfluorescence in the buccal cavity, legs, and feet, confirming that thetransfected ES cells contributed to these tissues.

FIG. 15 is a photograph of a chimeric chicken made with cES cellstransfected with CX/GFP/Puro under fluorescent light (right panel) andwhite light (left panel). Green fluorescence in the bones and in cellsin the feather pulp of the emerging primary wing feathers can beobserved, conforming that the transfected ES cells contributed to thesetissues.

FIG. 16 is a photograph of a chimeric chicken made with cES cellstransfected with CX/GFP/Puro under fluorescent light (left panel) andwhite light (right panel). Green fluorescencean be observed in theintestinal tissue and in breast muscle, confirming that the transfectedES cells contributed to these tissues.

FIG. 17 is a photograph of a chimeric chicken made with cES cellstransfected with CX/GFP/Puro under fluorescent light (left panel) andwhite light (right panel). Green fluorescence can be observed in the legmuscles, confirming that the transfected ES cells contributed to thesetissues.

FIG. 18 is a photograph of a chimeric chicken made with cES cellstransfected with CX/GFP/Puro under fluorescent light (left panel) andwhite light (right panel). Green fluorescence can be observed in thepancreas, confirming that the ES cells contributed to this tissue.

FIG. 19 shows the correlation between estimates of chimerism derived byscoring the level of fluorescence in skin and comb tissues and derivedby scoring the extent of black pigmentation of the chick at hatch.

FIG. 20 is a photograph of the allantois of a chimeric embryo made withcES cells transfected with CX/GFP/Puro under fluorescent light (leftpanel) and white light (right panel). Green fluorescence can be observedin the allantois, demonstrating that ES cells contributed to thistissue.

FIG. 21 is a summary of FACS data from chimeras made withnon-transfected cells (control), chimeras made with ES cells that weretransfected with CX/GFP/Puro and estimated to contain <5% donor-derivedcells by feather pigmentation, chimeras made with ES cells that weretransfected with CX/GFP/Puro and estimated to contain >5% and <10%donor-derived cells by feather pigmentation, chimeras made with ES cellsthat were transfected with CX/GFP/Puro and estimated to contain >10% and<30% donor-derived cells by feather pigmentation, chimeras made with EScells that were transfected with CX/GFP/Puro and estimated tocontain >30 and <75% donor-derived cells by feather pigmentation, andchimeras made with ES cells that were transfected with CX/GFP/Puro andestimated to contain >75% donor-derived cells by feather pigmentation.Cells were prepared as described in the text from liver, brain, andmuscle. The mean number of fluorescent cells detected above theautofluorescent threshold is shown in the left panel; the mean number offluorescent cells detected above the M2 threshold, which is an order ofmagnitude higher than the autofluorescent threshold is shown in theright panel.

FIG. 22 is an example of a FACS analysis of cells prepared from thebrain of a chimera made with non-transfected ES cells (upper panel) andchimeras that were made with cells transfected with CX/GFP/Puro (lowerpanel). Substantially all of the cells from the chimera made withtransfected cells display a level of fluorescence that is above that ofthe cells from the control bird indicating that the contribution of thedonor ES cells to the brain tissue of the chimera is extensive.

FIG. 23 is an example of a FACS analysis of cells prepared from legmuscles of a chimera that was made with cells transfected withCX/GFP/Puro. The right leg of this chimera was green to the naked eyeindicating substantial contribution to this tissue. A preparation ofcells from the fluorescent leg muscle was shown to contain primarilyfluorescing cells by FACS analysis (lower panel). The left leg of thechimera was normal in color and a preparation of these cells was shownto contain non-fluorescing cells by FACS analysis. These data show thatchimerism in tissues within the same animal may receive differentcontributions from donor ES cells.

DETAILED DESCRIPTION OF INVENTION

Pursuant to this invention, chicken ES cell lines are derived from stageX embryos that have a large nucleus and contain a prominent nucleolus(FIG. 1). These cells are confirmed to be chicken embryonic stem (cES)cells by morphology after long-term culturing and to yield chimeras wheninjected into recipient embryos. Moreover, the ES cells enable a highdegree of contribution to somatic tissues as determined by extensivefeather chimerism. Still further, these embryonic stem cells aredemonstrated to be transfected with transgenes carrying DNA encoding anexogenous protein. The ES cells stably integrate the transgene andexpress the transgene to enable selection of transformed cells. Thesetransformed cells are capable of forming chimeras wherein an exogenousprotein encoded by the transgene is present in the selected tissues ofthe chimera. Cells derived from the chimera express the exogenousprotein encoded by the transgene. In a particularly preferredembodiment, an exogenous proteins encoded by the transgene is expressedin a specific tissue or tissue types according to the protein encoded bythe transgene. Embryonic stem cell progeny are derivatives of ES cellsthat differentiate into non-ES cell phenotypes after introduction of theES cells into recipient embryos and the formation of a chimera. Atransgenic chicken is the progeny of a chimera which has been producedfrom chicken ES cells carrying a transgene which is stably integratedinto the genome when cells derived from the transgenic ES cells haveincorporated into the germline.

Broad expression of a transgene in somatic tissue is demonstrated byexpression in extraembryonic and somatic tissues. Analysis of theprotein content of egss of transgenic animals demonstrates selectiveexpression in the egg white of transgene-encoded exogenous proteinsobtained from the transgenes and using the techniques of the presentinvention. Tissue specific expression is demonstrated by expression thatis substantially confined to one organ, tissue, or cell type.

EXAMPLE 1 Derivation of Chicken Embryonic Stem Cells (cES Cells)

Chicken ES cells were derived from one of two crosses: Barred Rock XBarred Rock or Barred Rock X Rhode Island Red. These breeds wereselected to obtain a feather marker when testing the developmentalpotential of cES cells. The cES cells are injected into White Leghornembryos, which are homozygous dominant at the dominant, white locus.Chimeric chickens resulting from injection of these ES cells displayblack feathers from the cES cells and white feathers from the recipientembryo.

Initial establishment of the cES cell culture was initiated according tothe protocol described in U.S. Pat. No. 5,565,479. At stage X, theembryo is a small round disk, consisting of approximately 40,000-60,000cells, situated on the surface of the yolk. To retrieve the embryo apaper ring is put on the yolk membrane, exposing the embryo in themiddle. The yolk membrane is cut around the ring, which is then liftedoff the yolk. The embryo, attached to the ventral side of the ring, isplaced under the microscope and the area pellucida isolated from thearea opaca using a fine loop. TABLE 1 cES cell lines derived on eitherSTO feeder cells or a polyester insert in CES-80 medium. The cultureswere initiated from both single and pooled embryos. Donor Substrate usedto Cell line embryo derive cES cells Endpoint of cell line 009 pooledSTO cultured for 3 months, injected & cryopreserved 029 pooled insertcultured for over 3 months, injected & cryopreserved  31 pooled STOinjected at 4 days  36 pooled STO injected at 13 days  50 pooled STOgrown for over 8 months, injected & cryopreserved  63b pooled insertgrown for 3 months and cryopreserved  67I single insert injected at 45days of culture 307 pooled STO injected at 15 days and fixed forstaining 314 pooled STO cultured for over 3 months, injected &cryopreserved 317 pooled STO injected at 12 days and fixed for staining324A single insert cultured for over 6 months and injected 328 singleinsert cultured for over 6 months, injected & cryopreserved 329 singleinsert cultured for 5 months, injected & cryopreserved 330 single insertcultured for 3 months and cryopreserved 331 single 24 w insert culturedfor over 3 months and terminated 332 single 96 w STO cultured for 3months and cryopreserved 333 single 12 w insert cultured for over 3months and terminated 334 single 12 w insert cultured for over 3 monthsand terminated 335 single 96 w insert cultured for over 3 months andterminated

Embryos are dispersed mechanically into a single cell suspension andseeded on a confluent feeder layer of mitotically inactivated STO cellsat a concentration of 3×10⁴ cells/cm². The cES culture medium consistsof DMEM (20% fresh medium and 80% conditioned medium) supplemented with10% FCS, 1% pen/strep; 2 mM glutamine, 1 mM pyruvate, 1× nucleosides, 1×non-essential amino acids and 0.1 mM β-mercaptoethanol. Before use, theDMEM medium is conditioned on Buffalo Rat Liver (BRL) cells. Briefly,after BRL cells are grown to confluency, DMEM containing 5% serum isadded and conditioned for three days. The medium is removed and a newbatch of medium conditioned for three days and repeated. The threebatches are combined and used to make the cES medium. Chicken ES cellsbecome visible 3-7 days after seeding of the blastodermal cells. ThesecES cells were morphologically similar to mES cells; the cells weresmall with a large nucleus and a pronounced nucleolus (See FIG. 1).

The growth characteristics of cES cells are different from mES cells,which grow in tight round colonies with smooth edges and individualcells that are difficult to distinguish. Chicken ES cells grow in singlelayer colonies with clearly visible individual cells. Tight colonies areoften the first sign of differentiation in a cES culture.

To test for markers of pluripotency of the cells that were derived inculture, the cells were fixed and stained with SSEA-1 1 (Solter, D. andB. B. Knowles, Proc. Natl. Acad. Sci, U.S.A. 75: 5565-5569, 1978),EMA-1, which recognize epitopes on primordial germ cells in mice andchickens (Hahnel, A. C. and E. M. Eddy, Gamete Research 15: 25-34, 1986)and alkaline phosphatase (AP) which is also expressed by pluripotentialcells. The results of these test, which are shown in FIG. 2, demonstratethat chicken ES cells express alkaline phosphatase and the antigensrecognized by SSEA-1 and EMA-1.

Although cES cells are visible after using the above protocol, suchcultures cannot be maintained longer than a few weeks. Severalmodifications in culture conditions were initiated, as discussed below,which led to the derivation of 19 cell lines (Table 1) of which 14 weretested for their developmental potential by injection into recipientembryos. Eleven of the 14 cell lines contributed to recipient embryos asdetermined by feather pigmentation (See Table 2 below). This protocolyields sustained cultures of pluripotent cells expressing an embryonicstem cell phenotype. At any point, the cells can be cryopreserved andwhen injected into compromised recipient embryos have the potential tosubstantially contribute to somatic tissues (See Examples 3 and 5below).

Table 2: Passage number and time in culture of embryonic stem cell linesderived from single or pooled embryos. Frequency and extent of somaticchimerism after injection of these cES cells into stage X recipients.TABLE 2 Passage number and time in culture of embryonic stem cell linesderived from single or pooled embryos. Frequency and extent of somaticchimerism after injection of these cES cells into stage X recipients.time in # of Extent of Cell Donor Passage culture embryos # % chimerism¹line embryo number (days) injected # chimeras analyzed chimeras (%)  31pooled 0 4 15 2 7 28.5 1-5 317 pooled 4 12 29 2 10 20 25-65  36 Pooled 113 24 1 5 20 15 307 pooled 4 15 21 1 6 17  5 330 single 6 33 11 3 8 25 5-50 63b pooled 11 72 36 4 21 19  1-10 67I single 3 45 28 0 15 0 — 324Asingle 10 65 25 0 15 0 — 009 pooled 20 61 27 0 9 0 — 329 single 3 15 318 17 47  3-75 329 6 25 30 9 19 47  3-95 329 6 28 26 1 12 8 23 329 11 4910 1 4 25 60 029 pooled 4 33 40 9 27 33  5-80 029 9 37 40 4 15 27  4-15328 Single 6 56 19 4 11 36 10-80 328 12 98 33 7 22 32  5-50 314 Pooled17 52 30 2 5 40  5-65 314 15-17 53 29 1 4 25 30 314 17 55 37 3 15 30 3-80 314 16 65 27 2 11 18  5-40 314 14 61 25 0 13 0 — 314 16 65 32 3 1421 10-60 314 20 61 30 4 5 80  4-50 314 21 67 30 2 11 18  5-15 314 21 718 0 2 0 —  50 pooled 7 53 35 7 23 30  4-65  50 14 106 36 3 21 14 10-30¹Extent of chimerism was determined by the proportion of black feathers.

As with the mouse, avian embryonic stem cells, which are sometimesreferred to as embryonic germ cells, are derived on a variety of feederlayers including STO, STO-snl and others that are readily available.Leukemia inhibitory Factor (LIF) produced by these feeders, and theaddition of fetal bovine serum contributes to the maintenance of EScells in an undifferentiated state. In a preferred embodiment of thisinvention, chicken ES cell cultures are initiated on a STO feeder layer.STO cells are grown to confluency, treated with 10 μg/ml mitomycin for3-4 hours, washed, trypsinized and seeded on gelatin coated dishes at4×10⁴ cells/cm². cES cells appear to grow more rapidly when the feederof STO cells are sparser. Reducing the STO feeder cell concentration tobetween 10³ and 10⁵, and preferably below 10⁴ cells/cm², facilitates thederivation and propagation of cES cells. However, when chicken embryonicfibroblasts and mouse primary fibroblasts are used as feeders, no cEScells were derived. Also, when previously established cES cells wereplated on these feeders, all of them differentiated within 1 week.

Growing cES cells on synthetic inserts, such as polymer membranes(Costar, Transwell type) in the absence of feeders avoids contaminationof the recipient embryo with feeder cells when the ES cells areinjected. As Table 3 and 4 show, culturing on inserts, instead of STOfeeders, facilitates the derivation of cES cells, and inserts can beused for initial derivation. However, after initially growing rapidly oninserts, the mitotic activity of the ES cells declines after 4-6 weeksof culture. To extend the culture the cells have to be transferred to afeeder of STO cells. TABLE 3 Establishment of cES cells from singleembryos on either inserts or a feeder of STO cells (10⁴ cells/cm²). # ofcultures # of cell lines Substrate started obtained STO feeder 56 3(5%)  insert 45 7 (16%)

TABLE 4 Establishment of cES cells from pooled embryos on either a STOfeeder or a synthetic insert. # of cultures # of cell lines Substratestarted obtained STO feeder 73 7 (9.5%) insert 17 2 (12%) 

The data in Tables 3 and 4 show that chicken embryonic feeder cells andmouse primary fetal fibroblasts do not support the derivation or theculture of cES cells. A feeder of STO cells supports derivation andgrowth but only when present in a limited concentration of between 10³and 10⁵ STO cells but preferably in the present embodiment at aconcentration of less than or appropriately 10⁴ cells/cm². A dense STOfeeder layer impairs the growth of cES cells, while the specifiedconcentration of STO cells provides factor(s) necessary for ES cellproliferation. When the cells are sustained over an extended cultureperiod and continue to express an embryonic stem cell phenotype, anddifferentiate into non-embryonic stem cell phenotypes in vivo, they arereferred to as “ES cell progeny.”

The cES cell culture medium consists of 80% conditioned medium andpreferably contains certain BRL conditioned medium with factorsnecessary for the derivation and growth of cES cells. At a concentrationof 50%, growth of the cES cells is not as reliable as in 80% conditionedmedium. When the percentage of conditioned medium is reduced to lessthan 50%, the growth of the cES cells is affected, as evidenced by anincrease in differentiated cells and, at a concentration of 30% or less,the cES cells differentiate within 1 week. This conditioned medium foundnecessary for the derivation and maintenance of cES cells does notmaintain mES but causes their differentiation.

Fetal bovine serum is a preferred component of the ES cell mediumaccording to the present invention and contains factors that keep cEScells in an undifferentiated state. However, serum is also known tocontain factors that induce differentiation. Commercially availableserum lots are routinely tested by users for their potential to keep EScells in an undifferentiated state. Serum used for cES cell cultures areknown to differ from serum used for mouse ES cell cultures. For example,serum used for the culture of mouse ES cells that is low in cytotoxinand hemoglobin concentration, which is known to maintain mouse ES cellsin an undifferentiated state, did not support the sustained growth ofchicken ES cells.

Therefore, serum to be used on chicken ES cells should not be tested onmouse ES cells to determine suitability as a media component, butinstead should be evaluated on chicken ES cells. To do so, chicken EScell cultures are divided into two and used to test each new batch ofserum. The new batch tested must clearly support the growth of chickenES cells when empirically tested.

Chicken chromosomal spreads require special evaluation techniquesdifferent than mice because the complex karyotype consisting of 10macrochromosomes, 66 micro-chromosomes and a pair of sex chromosomes (ZZin males and ZW in females). The long-term cES cells of the presentinvention shown in FIG. 3 were analyzed after 189 days in culture andbeing cyopreserved twice. Referring to FIG. 3, they exhibited a normalkaryotype with 10 macro chromosomes; 2 Z-chromosomes and 66microchromosomes.

Chicken ES cells are cryopreserved in 10% DMSO in medium. After thawingand injecting several cell lines into recipient embryos, somaticchimeras are obtained, indicating that the cES cells maintain theirdevelopmental potential during the cryopreservation process.

EXAMPLE 2 Injection of Chicken Embryonic Stem Cells into RecipientEmbryos

To permit access to the embryo in a freshly laid egg the shell must bebreeched, inevitably leading to a reduction in the hatch rate at the endof the 21-day incubation period. The convention was to cut a small hole(less than 10 mm diameter) in the side of the egg, through which theembryo was manipulated, and re-seal with tape, a glass cover slip, shellmembrane or a piece of shell. Though relatively simple to perform, this“windowing” method caused embryonic mortality between 70 and 100%.Improved access to the embryo and increased survival and hatchabilitycan be achieved if the embryo is transferred to surrogate eggshells forincubation to hatching using two different shells and a method (adaptedfrom Callebaut) (Callebaut, Poult. Sci 60: 723-725, 1981) and (Rowlett,K. and K. Simkiss, J. Exp. Biol. 143: 529-536, 1989), which arespecifically incorporated herein by reference with this technique, themean hatch rate is approximately 41% (range 23-70%) with 191 chickshatched from 469 cES-cell injected embryos.

Incubation of embryos following injection of donor ES cells intorecipient embryos can be divided into two parts comprising System A andSystem B as described below:

System A covers the first three days of post-oviposition development.Fertile eggs containing the recipient embryos are matched with eggs 3 to5 grams heavier. A 32 mm diameter window is cut at the pointed pole, thecontents removed and the recipient embryo on the yolk, together with thesurrounding albumen, is carefully transferred into the surrogate shell.

Cells are taken up in a sterile, finely tapered glass pipette connectedto a mouth aspirator fitted with a 2 micron filter. The opening of thepipette can be from 50 to 120 microns in diameter and possesses a 300spiked bevel. The embryo is visualized under low magnification and withblue light. Chicken ES cells are trypsinized into a single cellsuspension and between approximately 2,000 and 26,000 cells andpreferably about 20,000 cells, are injected into an embryo. The cellsare gently expelled into the space either below or above the embryo,i.e. into the sub-embryonic cavity or between the apical surface of thearea pellucida and the perivitelline layer (yolk membrane) [Rob:preferably into the sub-germinal cavity]. Extra albumen collected fromfresh fertile eggs is added and the shell sealed with Saran Wrap plasticfilm.

System B covers the period from day three to hatching. At day three ofincubation the embryo has reached around stage 17 (H&H). Water has beentransported from the albumen to the sub-embryonic cavity, causing theyolk to enlarge and become very fragile. The contents of the System Ashell are very carefully transferred to a second surrogate shell(usually a turkey egg) 30 to 35 grams heavier than the original egg.Penicillin and streptomycin are added to prevent bacterial contaminationand the 38 to 42 mm window in the blunt pole is sealed with plasticfilm. This larger shell allows for an artificial airspace. At day 18 to19 of incubation the embryo cultures are transferred to tabletophatchers for close observation. As lung ventilation becomes established,holes are periodically made in the plastic film to allow ambient airinto the airspace. Approximately 6-12 hours before hatching the film isreplaced with a small petri dish, which the chick can easily push asideduring hatching.

For incubation, conventional temperature (37.5 to 38° C.) and relativehumidity (50 to 60%) are maintained for the embryos in surrogate shells,but periodic egg rocking, which is normally hourly and through 9.0degrees, has to be modified to ensure good survival. In System A rockingis through 90° every 4 to 5 minutes; in System B it is through 40 to 60°every 40 to 45 minutes. In both systems the speed of rocking ismaintained at 15 to 200 per minute.

The contribution of cES cells to chimeras is improved if the recipientembryo is prepared by (1) irradiated by exposure to 660 rads of gammairradiation (2) altered by mechanically removing approximately 1000cells from the center of the embryo, or by combining (1) and (2) abovebefore the injection of the cES cells. Referring to Table 5,contribution of cES cells to the somatic tissues increased substantiallywhen recipient embryos were compromised, either by removing cells fromthe center of the recipient embryo or by exposure to irradiation. Whenthe recipient embryos are compromised by a combination of irradiationand mechanical removal of the cells, the contribution of the ES cells isincreased further, even though the cES cells had been in culture forlonger periods of time. Some of the resulting chimeric chicks areindistinguishable from pure Barred Rock chicks (FIG. 4). As the data inTable 5 show, chimerism rates as well as the extent of chimerism perembryo increases after compromising the recipient embryo. TABLE 5Frequency of somatic chimerism after injection of cES cells intorecipient embryos that were compromised by different methods. Extent # #Embryos Frequency feather Treatment to compromise Cell Time cells # &chicks of chimerism the recipient embryo lines in culture Chimerasevaluated chimerism % (%) None 14 4-106 days 83 347 24 26 Mechanicalremoval of cells 1 6 months 34 63 54 20 Irradiation 1 6-7 months 56 9559 29 Irradiation &Mechanical 1 7-8 months 52 59 88 49 removal of cells

Recipient embryos substantially younger than stage X may also be used toproduce chimeras using ES cell as the donor. Early stage recipientembryos are retrieved by injecting the hens with oxytocin to inducepremature oviposition and fertile eggs are retrieved at stages VII toIX.

Alternatively, the retrieval of embryos from the magnum region of theoviduct provides access to stage I to VI embryos, consisting ofapproximately 4-250 cells, and enables the development of chimeras fromall embryonic stages as potential recipient embryos.

EXAMPLE 3 Somatic Chimeras from Chicken Embryonic Stem Cells (cES)

To demonstrate that pluripotency of the cES cells of the invention, cEScells are injected into White Leghorn recipient embryos. In the firstround of experiments, a total of 14 cell lines in 28 experiments areinjected into stage X recipient embryos (See Table 2). The cES cellshave been propagated in culture between 4 and 106 days and some lineshad been cryopreserved. Chicken ES cells are lightly trypsinized,resulting in small clumps of cES cells, and resuspended in DMEMsupplemented with 25 mM HEPES+10% fetal calf serum. Three to five μl ofthe cell suspension, containing between 2000-5000 cells, are injectedinto the subgerminal cavity of the recipient embryos. All embryos thatdeveloped feathers are analyzed and twenty four percent of embryos(83/347) are chimeric as determined by feather color. Feather chimerasare obtained from 11/14 cell lines. The extent of chimerism varied from1%-95% with a mean extent of 25.9% (SD=20.4).

Table 2 illustrates the variance in the somatic chimerism betweenexperiments performed within and between cell lines. Examples of thecontribution of ES cells to chimeras is shown in FIGS. 4 and 5. In FIG.4, two chicks are chimeras and two are Barred Rocks; it is apparent thatthere are no phenotypic differences between these chicks indicating thatthe contribution of ES cells to the chimera is extensive, particularlyin the ectodermally derived lineages. In FIG. 5, the chimeras on theleft have relatively low levels of contribution from ES cells whereasthose on the right have intermediate contributions.

EXAMPLE 4 Transfection of cES Cells by Lipofection and Electroporation

Referring to Table 6, an appropriate amount of DNA compatible with thesize of the well being transfected is diluted in media absent of serumor antibiotics. The appropriate volume of Superfect (Stratagene) isadded and mixed with the DNA, and the reaction is allowed to occur for5-10 minutes. The media is removed and the wells to be transfected arewashed with a Ca/Mg free salt solution. The appropriate volume of media,which can contain serum and antibiotics, is added to the DNA/superfectmixture. The plates are incubated for 2-3 hours at 37 C. When theincubation is completed, the Superfect is removed by washing the cells1-2× and fresh culture media is added. TABLE 6 Conditions fortransfection of chicken ES cells using Superfect. Volume Time to Volumeof of media Total form media used to dilute amount of ul complex addedto Incubation Plate Size DNA DNA Superfect (min) complex time  96 well30 ul 1  5 ul 5-10 150 2-3 hrs  48 well 50 ul 1.5  9 ul 5-10 250 ul 2-3hrs  24 well 60 ul 2 10 ul 5-10 350 ul 2-3 hrs  12 well 75 ul 3 15 ul5-10 400 ul 2-3 hrs  6 well 100 ul  4 20 ul 5-10 600 ul 2-3 hrs  60 mm150 ul  10 50 ul 5-10 1000 ul 2-3 hrs 100 mm 300 ul  20 120 ul  5-103000 ul 2-3 hrs

A petri-pulser is used to electroporate cES cells that are attached tothe plate in a 35 mm diameter well. The media is removed and the well iswashed with a salt solution without Ca⁺⁺ and Mg⁺⁺. One ml ofelectroporation solution is added to the well. DNA is added and themedia is gently mixed. The petri-pulser is lowered onto the bottom ofthe well and an electrical current is delivered. (Voltage preferablyvaries from 100-500 V/cm and the pulse length can be from 12-16 msec).The petri-pulser is removed and the electroporated well is allowed tostand for 10 minutes at room temperature. After 10 minutes, 2 mls ofmedia is added and the dish is returned to the incubator.

To transfect cells in suspension, media is removed and cells are washedwith a Ca/Mg free salt solution. Tryspin with EDTA is added to obtain asingle cell suspension. Cells are washed, centrifuged and resuspended ina correctional electroporation buffer solution such as PBS. The ES cellsuspension is placed into a sterile cuvette, and DNA added (minimumconcentration of 1 mg/ml) to the cell suspension and mixed by pipettingup and down. The cells are electroporated and allowed to sit at RT for10 minutes. Cells are removed from cuvette and distributed to previouslyprepared wells/dishes. Cells are placed in an incubator and evaluated ortransient transfection 24-48 hours after electroporation. Selection ofantibiotic resistant cells may also be started by including anantibiotic such as puromycin in the culture medium.

In a preferred embodiment, the concentration of puromycin required forselecting transfected cells is calculated as a titration kill curve.Titration kill curves for chicken embryonic stem cells are establishedby exposing cells in culture to puromycin concentrations varying from0.0 to 1.0 μg/ml for 10 days (Table 7) and neomycin concentrationsvarying from 0.0 to 200 μg/ml (Table 8). The medium is changed every 2days and fresh puromycin or neomycin is added. When exposed to aconcentration of 0.3 μg/ml puromycin, ES cells were absent from allwells after 3 changes of medium with fresh puromycin over a six dayperiod (see Table 7). Puromycin concentrations of 0.3-1.0 μg/ml are usedfor selection of the transfected cultures. Neomycin concentrations over40 μg/ml eliminated all cES cells within 7 days (Table 8).

After 10 days of selection, cES cells colonies are visible and can bepicked for further expansion.

Table 7: Morphology of cES cells after exposure of variousconcentrations of puromycin and different lengths of time (days afteraddition of puromycin). TABLE 7 Morphology of cES cells after exposureof various concentrations of puromycin and different lengths of time(days after addition of puromycin). Puromycin conc. Time under selection(days) (μg/ml) 1 2 3 4 5 6 7 8 9 10 0.0 ES ES ES ES ES ES ES ES ES ES0.1 ES ES ES ES ES ES ES ES ES ES 0.2 ES ES ES ES ES ES ES ES ES ES 0.25ES ES ES ES ES diff diff diff/ diff/ diff/ gone gone gone 0.3 ES ES diffdiff/ diff/ gone gone gone gone gone gone gone 0.4 ES diff gone gonegone gone gone gone gone gone 0.5 diff gone Gone gone gone gone gonegone gone gone 0.6 diff gone gone gone gone gone gone gone gone gone 0.7diff gone gone gone gone gone gone gone gone gone 0.8 gone gone gonegone gone gone gone gone gone goneES: ES cells are present.diff: ES cells are differentiated.gone: no morphologically recognizable cells are present

TABLE 8 Morphology of cES cells after exposure of various concentrationsof neomycin and different lengths of time (days after addition ofneomycin). Neomycin conc. Time under selection (days) (μg/ml) 1 2 3 4 56 7 8 9 10 0.0 ES ES ES ES ES ES ES ES ES ES 10 ES ES ES ES ES ES ES ESES ES 20 ES ES ES ES ES ES ES ES ES ES 30 ES ES ES ES ES ES ES/DiffES/diff Diff Diff/ gone 40 ES ES ES ES ES/Diff Diff/ dead gone gone gonedead 50 ES ES ES ES/Diff ES/Diff Diff/ Dead/ gone gone gone dead gone 60ES ES ES gone gone gone gone gone gone gone 100 ES/Diff Diff dead gonegone gone gone gone gone gone 150 dead dead gone gone gone gone gonegone gone 200 dead gone gone gone gone gone gone gone gone

Section of transfected chicken ES cells and their identification inchimeras requires that the transgene confer a selective advantage to thecells in culture (e.g. resistance to puromycin in the medium) and thatit produce an identifiable gene product in the cells in the chimerawhich are derived from the ES cells. This can be accomplished usingpCX/GFP/Puro which provides resistance to puromycin in cES cells andproduces a green fluorescent protein (GFP) in most, if not all,donor-derived cells in chimeras.

Referring to FIG. 6, PCX/GFP/Puro (FIG. 6) was produced in three cloningsteps involving two intermediates before make the final pCX/GFP/Puroplasmid. In step 1, the PGK-driven Puromycin resistant gene cassette(1.5 Kb) was released from pKO SelectPuro (Stratagene) by Asc Idigestion. The fragment was then blunted and Kpn I linkers were added.The resulting fragment (GFP/Puro) was inserted into the correspondingKpn I site of pMIEM (courtesy of Jim Petitte NCSU), a GFP expressionversion derived from LacZ expression pMIWZ, see Cell Diff and Dev. 29:181-186 (1990) to produce the first intermediate (pGFP/Pro). ThePGK-Puro cassette was in same transcription orientation as GFP(determined by BamH I and Sty I digestion). In step 2, the GFP/Puroexpression cassette (2.5 Kb) was released from pGFP/Puro by BamH I andEcoR I double digestion. The resulting fragment was inserted into theBamH I and EcoR I sites of pUC18 (Invitrogen). It contains 5′ uniquesites, Hind III, Pst I and Sal I. The resulting plasmid pUC18/GFP/Purowas verified by a BamH I, EcoR I, and Not I triple digestion. In thethird step, the Cx promoter including 384 bp CMV-IE enhancer, 1.3 kbchicken beta-actin promoter and portion of 1^(st) intron was releasedfrom pCX-EGFP (Masahito, I. et al., FEBS Letters 375: 125-128, 1995) bySal I and EcoR I digestion. A 3′ EcoR I (null)-Xmn I-BamH I linker wasattached to the fragment and it was inserted into the Sal I and BamH Isites of pUC18/GFP/Puro. The plasmid pCX/GFP/Puro (FIG. 6) was verifiedby a BamH I and Pst I double digestion. pCX/GFP/Puro DNA can belinearized by Sca I digestion for transfection into cES cells.

Transfection and selection of ES cells using the procedures describedabove produced a population of cells that would grow in the presence of0.5 ug of puromycin. These cells exhibited green fluorescence whenexamined by conventional fluorescence microscopy (See FIG. 7). Whenpreparations of the ES cells are examined by fluorescence activated cellsorting, it is evident that essentially all of the cells carry andexpress the transgene (See FIG. 8). Southern analysis of DNA from thetransfected ES cell lines TB01 and TB09 that was digested with BamH1,EcoRI or both restriction endonucleases revealed the transgene in DNAfragments of various sizes, providing evidence that the transgene isintegrated into the genome (See FIG. 9).

The CX/GFP/Puro construct demonstrates that transgenes of at least 4.5kb can be inserted into chimeric chickens. Using the cES cells describedherein, chicken ES cells can be transfected with different or largerconstructs. Depending on the design of the transgene, DNA encoding anexogenous protein may be widely detected in somatic cells of theresulting chimeric or transgenic animal, i.e., the endoderm, mesoderm,ectoderm, and extra embryonic tissue, or may be designed to expresssignificant levels of an exogenous protein only in a selected tissue.The protein is encoded by DNA contained in a tissue specific transgeneconstruct. The transgene construct may be comprised of genetic elementsderived from the genome of the host organism and selected on the basisof known expression, or patterns of expression, of a protein in aselected tissue. For expression in a particular tissue, a gene encodinga protein that is normally expressed, and usually highly expressed inthe selected tissue, is selected and regulatory elements from the geneare chosen to drive expression of the exogenous protein. When combinedwith DNA coding sequences for the exogenous protein, other regulatoryelements, selectable markers, etc. the transgene yields preferentialexpression of the exogenous protein in the selected tissue. Preferentialexpression in a specific tissue type may be defined as 3 to 4 orders ofmagnitude greater expression in the selected tissue in comparison tonon-selected tissues.

For tissue specific protein expression in a transgenic bird, the tissuespecific expression is preferably directed to a region of the oviductincluding the magnum, isthmus, shell gland, or infundibulum. Theselection of the individual tissue for protein expression depends on thetype of protein to be expressed. The magnum contains the tubular glandcells that express the predominant proteins of the egg white, while theisthmus contains cells that express the shell membrane. Soluble proteinexpression is preferably directed to the tubular gland cells of themagnum of the oviduct by selecting regulatory sequences, usuallycomprising a promoter, from genes expressing egg white proteins,preferably ovalbumin, but including ovotransferrin, ovomucoid, lysozyme,ovoglobulin G2, ovoglobulin G3, ovoinhibitor, cystatin, ovoglycoprotein,ovoflavoprotein, ovomacroglobulin, and avidin. Selective expression inthe tubular gland cells, to the exclusion of expression in theepithelial cells is demonstrated below and termed preferentialexpression to distinguish selectivity between the two cell types.

In the following example, a transgene containing an endogenous egg whiteregulatory sequence comprising a promoter together with an exogenousimmunoglobulin locus is constructed to yield tissue specific antibodyexpression in the tubular gland cells of the oviduct. The antibodymolecules so expressed are then deposited in the egg white of atransgenic chicken. In this embodiment, antibodies encoded by anyrearranged immunoglobulin gene are expressed specifically in tissuecomprising the tubular gland cells of the magnum region of the oviductand can be isolated from the whites of eggs. The rearrangedimmunoglobulin gene encoding a monoclonal antibody is preferentiallyexpressed in the oviduct to the substantial exclusion of expression inother tissues, although expression in other tissues may exist abovedetectable levels.

In this embodiment, the monoclonal antibody cassette under the controlof the ovalbumin regulatory sequences is comprised of at least 3.4 kband preferably at least approximately 7.5 kb of the 5′ regulatorysequence and may include 15 kb or more of the 3′ regulatory sequence.Preferably, the construct includes regions of the ovalbumin geneflanking both the 5′ and 3′ ends of the exogenous antibody coding regionalthough a large enough segment of the endogenous promoter sequence ofthe 5′ flanking region may avoid the need for a 3′ flanking region. Thecoding regions of both the heavy and light chains of the antibody areprovided in the transgene and include the variable, diversity, joiningand constant regions of the selected isotype. In a preferred embodiment,the antibody is encoded by an immunoglobulin gene that ischaracteristically human and contains at least a human heavy chain.Also, the isotype is preferably IgG, and most preferably IgG1.

A preferred transgene construct for an ovalbumin derived monoclonalantibody construct is provided in FIG. 10A. This transgene construct isdesignated Ov7.5 and has approximately 7.5 kb of an egg white regulatorysequence comprising a promoter, in this specific embodiment theovalbumin promoter, flanking the MAb coding at the 5′ end sequence and15 kb of the promoter sequence 3′ of the coding region. The codingregions comprise the variable regions for both the light chain and heavychain, J-C intron sequences, the kappa light chain constant region, anIRES sequence and the gamma 1 isotype heavy chain constant regions. The3′ end of the construct comprises a GFP gene and a selectable marker, inthis case a puromycin resistance gene driven by the CX promoterdescribed herein. The lengths of the ovalbumin promoter sequence both 3′and 5′ of the monoclonal antibody coding region are examples only andanalogous constructs may include 25-100 kb or more of the 5′ sequence aswell as varying lengths in the 3′ sequence. Those of ordinary skill inthe art will appreciate that the GFP marker is present only fordetection in physiological specimens and can be removed withoutdeparting from the utility of the transgene. The puromycin resistancemarker can be substituted with any marker that provides the ability toselect embryonic stem cells that are successfully transformed with thetransgene. Several types of analogous selectable marker are well knownin the art and can be used essentially interchangeably with thepuromycin resistance gene of this embodiment.

As noted above, this monoclonal antibody is only one example of severaltypes of monoclonal antibody products that may be expressed using thetransgene constructs of the invention. Moreover, monoclonal antibodiesas a class of proteins are only one example of many classes of proteinproducts that may be expressed in tissue-specific fashion pursuant tothe methods and techniques described herein.

Referring to FIG. 10B, a section of the magnum of two-week old chimerasin which expression of the Ov 7.5 transgene was induced by estrogeninjection shows tissue specific expression of the anti-dansyl monoclonalantibody producing cells derived from the transformed embryonic stemcell express GFP, which shows as green in the top left panel of FIG.10B, confirm contribution by the embryonic stem cell transformed withthe Ov 7.5 transgene. Referring to the bottom left panel of FIG. 10B,the monoclonal antibody stains red in the tubular gland cells, while theepithelial cells, which are also derived from the donor embryonic stemcell, stain green and do not stain red. This difference in stainingdemonstrates that expression of the construct is tissue specific andselected by the content of the transgene for the specific tissue type.While the example below demonstrates tissue specific expression in thetubular gland cells of the oviduct, the demonstration of expressionacross all cell and tissue types demonstrates that each or any tissuetype could be chosen for tissue- or cell-specific expression bycorresponding selection of the components of the transgene construct,and e.g. the promoter or other regulatory elements.

In the top right panel of FIG. 10B, all of the cell types are shown byDAPI staining. In the bottom right panel, the stains are overlaid todemonstrate that only donor-derived tubular gland cells express themonoclonal antibody, while recipient-derived cells and donor-derivedepithelial cells do not express the monoclonal antibody. FIGS. 10C and10D are RT-PCR analyses showing that the heavy chain and light chain,respectively, of the anti-dansyl monoclonal antibody are selectivelyexpressed in only the oviduct tissue of 3 or 5 chimeras and not in thebrain, gut, pancreas or muscle of these chimeras above levels detectedby RT-PCR.

To demonstrate that a human IgG isotype monoclonal antibody could beselectively expressed and the protein deposited is egg white, a total of18 chimeric females were produced by injecting cES cells carrying theOv7.5 construct. Six chimeras from this group were used for testingtransgene expression by early estrogen induction. The remaining 12chimeric females were reared to sexual maturity for egg collection. Ninechimeric females commenced laying at 17-22 weeks of age and one of thesechimeras laid sporadically. Three of the chimeric females had not laideggs at 35 weeks of age and at autopsy it was evident that their gonadwas masculized by the presence of male ES cell derived tissue.

Eggs from the nine egg laying chimeras were collected and representativeegg white samples were prepared by ammonium sulfate precipitation andanalyzed by ELISA. The microtiter plates were coated with a goatanti-human IgG antibody and the presence of human IgG MAb in the eggwhite samples was revealed by either a labeled goat anti-human IgG (γchain specific) antibody for heavy chain and/or a labeled goatanti-human kappa (κ chain specific) antibody for light chain. Thestandard curves were established with purified human Igγ1, κproteins.The sensitivity of the ELISA was 0.8 ng/ml. Egg white samples fromnon-transgenic White Leghorn hens were used as negative controls. Nohuman IgG MAb deposition was detectable in eggs from non-transgenicWhite Leghorn hens (4 eggs) or from 6 chimeric hens (8 eggs from hen#OV11-17, 8 eggs from hen #OV11-53, 6 eggs from hen #OV11-73, 6 eggsfrom hen #OV11-88, 4 eggs from hen #OV12-97 and 5 eggs from hen#OV13-13).

Human IgG MAb deposition were detected in eggs from 3 different chimerichens (˜1.4-6.3 ng/ml for eggs from hen #OV11-13, ˜2.0-2.9 ng/ml for eggsfrom hen #OV11-37 and ˜2.9-10.8 ng/ml for eggs from hen #OV11-43 asdetermined by ELISA for IgH). The concentration of human IgG MAb inrepresentative eggs is summarized in Table 9. The concentration of humanIgG MAb in eggs determined by ELISA for IgL was consistently lower thanthat determined by ELISA for IgH. In general, the concentrationdetermined by IgL was 60% of the concentration determined by IgH(compare columns 3 and 5 in Table 9). The difference was also present inspiked samples made with purified human Igγ1, κ proteins. TABLE 1Deposition of human IgG MAb in eggs from chimeras Human IgG CorrectedHuman IgG level by IgH IgG value in level by IgL ELISA egg white ELISAEgg ID Date laid (ng/ml)* (ng/ml)** (ng/ml)* OV11-43-9 Nov. 12, 2002 7.610.8 4.6 OV11-43-10 Nov. 13, 2002 5.2 7.4 3.4 OV11-43-11 Nov. 14, 20026.8 9.7 4.2 OV11-43-13 Nov. 17, 2002 5.4 7.7 3.4 OV11-43-14 Nov. 18,2002 4.6 6.6 3.2 OV11-43-25 Nov. 30, 2002 3.0 4.3 1.8 OV11-43-26 Dec.01, 2002 2.0 2.9 1.2 OV11-43-33 Dec. 08, 2002 7.0 10.0 4.2 OV11-13-1Nov. 24, 2002 3.6 5.1 2.4 OV11-13-2 Nov. 26, 2002 4.4 6.3 2.6 OV11-13-3Dec. 2, 2002 3.8 5.4 2.4 OV11-13-4 Dec. 10, 2002 2.6 3.7 1.4 OV11-13-5Dec. 11, 2002 3.6 5.1 2.2 OV11-13-11 Dec. 17, 2002 3.2 4.6 2.2OV11-13-37 Jan. 27, 2003 1.0 1.4 ˜0.6 OV11-37-1 Dec. 08, 2002 2.0 2.91.2 OV11-37-2 Dec. 11, 2002 2.0 2.9 1.2 OV11-37-3 Jan. 06, 2003 1.4 2.0˜0.6*Some egg white samples were prepared and assayed multiple times and thevalues were averaged.**The recovery for sample preparation was estimated at 50-70% usingnegative control egg white samples spiked with known amount of purifiedhuman Igγ1, κ proteins. The IgG value given in Column 4 was correctedbased on a recovery of 70% for sample preparation to provide aconservative estimate of the concentration of human Ig in egg white.

The concentration of human IgG MAb proteins in blood samples from hens#OV11-13, #OV11-37 (which deposited the human Ig in their eggs) and aWhite Leghorn was less than the sensitivity of the assay (0.8 ng/ml).These data are consistent with the absence of ectopic expression ofhuman Ig in chimeric chickens that was observed using RT-PCR to evaluatethe presence of the human Ig transcripts in gut, brain, pancreas andmuscle in estrogen induced chimeric chicks (FIGS. TI 10C and D). TheOv7.5 construct, therefore, appears to deliver tissue specific,hormonally induced and developmentally regulated gene expression intransgenic chimeric hens. Furthermore, the protein appears to beexported from the tubular gland cells in the magnum and deposited in eggwhite.

The ovalbumin-derived tissue specific protein expression transgenes wereconstructed as follows:

A chicken genomic BAC library (Crooijmans, R. P. et al., Mamm. Genome11: 360-363, 2000), (Texas A & M BAC Center) is screened to isolate aregion of 46 Kb in the ovalbumin locus. Two different vectors wereconstructed having different fragments of the ovalbumin promoter located5′ of the MAb coding region: (1) Ov7.5MAb-dns: A 42 Kb expression vectorcontains 9.2 Kb 5′ sequences from the ovalbumin gene (including 7.5 Kbpromoter) and 15.5 Kb 3′ flanking sequences (FIG. 10A). This 42 kbexpression vector contains 9.2 kb of 5′ sequence from the ovalbumin gene(including 7.5 kb promoter) and 15.5 kb 3′ flanking sequences. Abicistronic monoclonal antibody cassette encodes the light chain, anIRES and the heavy chain of an anti-dansyl antibody. (2) Ov15MAb-dns: A49 Kb expression vector contains 16.8 Kb 5′ sequences from the ovalbumingene (including 15 Kb promoter) and 15.5 Kb 3′ flanking sequences (notshown). The 49 kb expression vector contains 16.8 kb of 5′ sequence fromthe ovalalbumin gene (including 15 kb promoter) and 15.5 kb3′ flankingsequences. The monoclonal antibody cassette is identical in bothconstructs.

As noted above, the gene to be expressed in both vectors is amouse-human hybrid anti-dansyl monoclonal antibody (MAbdns). TheCxEGFP/CxPuro cassette is cloned in the most 3′ end to allow selectionwith puromycin for stable transfection in cES cells and easyidentification of transfected cells in chimeras. Both constructs arelinearized and purified before transfection into cES cells.Transfections of cES cells are performed with Ov7.5MAbdns and Ov15MAbusing either SuperFect (Stratagene) or petri-pulser electroporation.After selection with puromycin, 6 resistant clones are picked formolecular analysis. The presence of the transgene is confirmed by PCRwith primers located in the MAbdns cassette, in the GFP gene and in thePuro gene. Transfected ES cells are used to create transgenic orchimeric birds as described in detail below.

In yet another example, a very large transgene encoding part of theunrearranged human heavy chain immunoglobulin locus has been transfectedinto chicken ES cells. A 139 kilobase bacterial artificial chromosome(BAC) clone was co-transfected with the pCX-EGFP-CX-puro selectablemarker into cES cells by co-lipofection of circular BAC DNA and linearselectable marker DNA. The BAC clone contains a human genomic DNA insertfrom an unrearranged immunoglobulin heavy chain locus and contains themost 3′ variable region (V_(H)6-1), all the diversity (D) segments, allthe joining (J) segments, the Cmu and Cgamma constant regions, theJ-intronic enhancer, and all the intervening DNA between these elements.It also includes the human gene KIAA0125, a gene that encodes anon-translated RNA of unknown function that is found between V_(H)6-1and the D segment region. pCX-EGFP-CX-puro is a plasmid that containsthe Enhanced Green Fluorescent Protein (EGFP) gene driven by the CXpromoter (consisting of a cytomegalovirus enhancer and the chickenβ-actin promoter) and a puromycin resistance gene driven by the samepromoter. The cES cells transfected with this plasmid are greenfluorescent and resistant to the antibiotic puromycin. The presence ofthe unrearranged human heavy chain locus in the transfected ES cellsthat were growing in the presence of puromycin was examined by PCRanalysis of transgenes spread throughout the 139 kb construct. Theprimer sequences used in the PCR analysis were: VH6-1: V6-1FAGTGTCAGGGAGATGCCGTATTCA V6-1R ACTTCCCCTCACTGTGTCTCTTG D1-26: D1-26FGGGCGCCTGGGTGGATTCTGA D1-26R GTGGCCCCTAAACCTGAGTCTGCT D1-20: D1-20FCCCGAGCACCGTCCCCATTGA D1-20R GTGCCGGTGATCCCTGTCTTTCTG Cμ: Mu1FGCGGGAGTCGGCCACCATCACG Mu1R AGCACAGCCGCCGCCCCAGTAG Cδ: Delta1FTGGGGAGAGGAGAGCACAGT Delta1R GGCGGGCGTAGGGGTCAGC

cES cells are co-transfected with the selectable marker pCX-EGFP-CX-puroand the BAC CTD-2005N2, resulting in a cES cell line designated BAC-A.Genomic DNA is prepared and PCR performed using 5 different primer setscorresponding to markers along the length of the BAC clone. Thesemarkers are: V_(H)6-1 (24 kb from the 5′ end [relative to the humanheavy chain locus] of the human genomic insert), D1-26 (83 kb from theend), D1-20 (73 kb from the end), Cμ (˜108 kb from the end), and Cδ (120kb from the end). Only V_(H)6-1, Cμ, and D1-26 are shown but all gavesimilar results. As a control for amplification from the cES cellsample, a chicken β-actin PCR is also run. The samples are:

-   -   1. BAC-A cells;    -   2. Mouse STO cells used as a feeder layer for the cES cells        (negative control);    -   3. Barred Rock embryo DNA (the same strain a the parental cES        cell line, negative control);    -   4. Human genomic DNA (positive control);    -   5. cES cell medium (negative control).

As shown in FIG. 11, all segments of the transgene are present in thetransfected and selected ES cells.

EXAMPLE 5 Identification of Transfected Donor-Derived Cells in Chimeras

Transfected donor derived cells are detected in chimeras by a number ofmethods. For example, the transgene is detected by Southern analysis oftissue taken from chimeras that are identified by the presence of blackpigmentation in their feathers. Also, DNA is harvested from embryos orfrom comb tissue and digested with BamH1, EcoR1 or a combination ofthese endonucleases. When DNA from tissues in the chimeras are examinedby Southern analysis, bands that were identical to those seen in TB01,which was the donor cell used to make the chimeras, were evident afterdigestion with either BamH1, EcoR1 or a combination of these tworestriction endonucleases (FIG. 12), thus providing evidence that thechimera contains progeny of the ES cells that were introduced into therecipient embryo to form the chimera.

The presence of the transgene in tissues is detected by illuminating thechimeras with fluorescent lights, which shows that the transgene isexpressed in the eye and the beak (FIG. 13), the buccal cavity, legs andfeet (FIG. 14), and in the bones of the wing and in cells in the featherpulp (FIG. 15). Examination of the internal organs revealed thatdonor-derived ES cells had contributed to the intestinal tissues andbreast muscle (FIG. 16) and leg muscle (FIG. 17). In another bird,extensive contributions to the pancreas were observed (FIG. 18). Thesedata provide compelling evidence that the progeny of ES cells contributeto the ectoderm which gives rise to the beak, feathers and skin, to themesoderm which gives rise to muscle and bone and to the endoderm, whichgives rise to the pancreas.

Illumination of the bird with fluorescent light is used to score thebirds on a scale of 1 to 4 where 4 indicates that all of the visibleskin, eyes and comb are fluorescent. The fluorescent score wasdetermined by screening several areas of the skin with a fluorescentlamp and scoring the chicks on a scale of 0 to 4. A similar score isused to rate the extent of chimerism estimated by feather pigmentation.Feather chimerism was estimated as % of black down compared to down athatch. Approximately 25% of the down of a Barred Rock chick is white.The maximum score that is possible in a chimera derived completely fromBarred Rock derived ES cells is, therefore, 75%. The correlation betweenthe two which is described by the equation y=0.41×+0.25 where y is thefluorescence score and x is the extent of ES-cell derived featherpigmentation is shown in FIG. 19.

Examination of the chorioallantois indicated that cES cells alsocontributed to the extraembryonic tissues (See FIG. 20). These dataindicate that the pluripotentiality of chicken ES cells may be greaterthan that of murine ES cells, which do not contribute to thetrophectoderm.

In addition to examination of live tissues in chimeras, cells wereprepared from a variety of tissues and the proportion of ES-cell derivedfluorescing cells was determined. Tissue (muscle, liver or brain) wasremoved from chickens post-mortem, then rinsed with PBS to remove bloodor other fluids. The outer membrane of the tissue was dissected away andthe remaining sample was minced. Each tissue sample was transferred to amicrocentrifuge tube containing 1 ml of 1 mg/ml collagenase (Type IV,Sigma) in either DMEM (muscle and liver) or Liebovitz L15 medium (brain)without serum. These tubes were incubated in a 37° C. water bath for30-45 minutes (the tubes were inverted every 10-15 minutes to shake thesuspension) and then the tissues were dispersed into single cellsuspensions using a 100-200 ul pipette equipped with a disposableplastic tip and purified according to the tissue type. Allcentrifugation steps were executed in a microcentrifuge.

For the muscle cell suspension, the suspension was centrifuged for 5 minat 4000 rpm, the supernatant removed and replaced with 500 ul of DMEMcontaining 10% heat inactivated horse serum. The pellet was resuspended,and the tube centrifuged for a further 60 seconds at 2600 rpm, thesupernatant was then removed and filtered through 40 micron nylon mesh(sterile disposable, Falcon brand). A sample of the cell suspension wasinspected by microscopy at this stage to ensure correct cell morphologyand sufficient cell density for flow cytometry (sub optimal densitieswere corrected by dilution, or by pelleting and resuspension).

For the liver cell suspension, the suspension was filtered through40-micron nylon mesh, transferred to a clean microcentrifuge tube andpelleted as for muscle. The top layer of the pellet was carefullyremoved to a clean tube with complete DMEM (500 ul), taking care not todisturb the red blood cells which form a distinct and visible layer atthe bottom of the pellet. The cell suspension was inspected at thisstage and the cell density adjusted. If red blood cells were present inthe cell preparation they were removed with a lysis step (incubating thecells in 1 ml lysis buffer containing 130 mM Ammonium Chloride, 17 mMTris, 10 mM Sodium Bicarbonate for 5 minutes at room temperature).

For the brain cell suspension, the suspension was filtered and pelletedas described above. The pellet was resuspended in 250 ul completeLiebowitz (L-15) medium containing 10% horse serum and 6 g/l glucose,Percoll was added to produce a 50% solution (approx. 260-280 ul). Thesuspension was centrifuged at 3.5 k for 5 minutes, following which thetop layer of cells was carefully removed into a clean microcentrifugetube and diluted with at least 1 ml of L-15 medium. The cells werepelleted by centrifuging at 4000 rpm for 5 minutes and then wereresuspended in an appropriate volume of L-15.

A flow cytometric analysis of GFP expression was conducted in liver,brain and muscle cells from chimeras. Single cell suspensions weretransferred into polystyrene tubes and loaded into a flow cytometer, forwhich the operating parameters had been set to detect the particularcell type of the sample, and which was equipped to detect emitted lightat a green wavelength in response to an excitory laser beam. At eachanalysis, at least one group of samples from a non-transgenic chimerawas analyzed in order to set the baseline for fluorescence measurement(since the flow cytometer detects some autofluorescence from each cell).These are referred to as control samples in FIG. 21. The data producedby the flow cytometer included the number of cells detected (within theparameters specified) and summarized the fluorescence of the cells inthat population. Examples of the data from analyses of brain and muscletissue is shown in FIGS. 22 and 23, respectively. Data was collected forthe number of cells which exhibited a fluorescence intensity greaterthan the autofluorescent level set by the non-transgenic sample for eachtissue type (designated M1). A susbset of these data was collected forcells which exhibited a fluorescence intensity at least ten timesgreater than the autofluorescent level (designated M2).

Brain, liver and muscle (breast and leg) samples were removed fromtwenty six chickens, of which 18 were chimeras that had been produced byinjecting transgenic chicken ES cells carrying the GFP transgene intonon-transgenic White Leghorn recipient embryos, as described above. Theremaining 8 chickens were chimeras that were produced by injectingnon-transfected cES cells into non-transgenic White Leghorn recipientembryos. Male and female chickens were present in both the groups. Greenfluorescence was detected in brain-derived, liver-derived andmuscle-derived cells from the transgenic chimeras. The fluorescenceintensity and the ratio of fluorescent to non-fluorescent cells variedbetween birds and between tissue type. The tabulated results are shownin FIG. 21. In birds which had been awarded a low score fordonor-derived feather pigmentation, or for green skin when screened witha UV lamp prior to post-mortem analysis, the number of fluorescent cellsin the tissue samples was generally low or zero. Birds which scored wellfor these criteria (e.g. greater than 75% donor-derived feathers) werefound to have fluorescent cells in all three tissue sample types, andthis was the only group in which highly fluorescent (M2) cells werepresent in all three tissue types. Of the three tissue types, the numberof fluorescent cells derived from the liver was the lowest. Brain andmuscle-derived fluorescent cells were present in greater numbers and ina greater number of samples derived from the transgenic chimeras. Thedata is shown graphically in FIG. 19.

The data shows that transgenic cES cells, when injected into recipientstage X embryos, can thrive in the host and differentiate into liver,muscle and brain cells. Liver, muscle and brain are tissues that arederived from endoderm, mesoderm and ectoderm, respectively; thus cEScells may differentiate into the three major somatic cell lineages, fromwhich all other somatic cells are derived. Further, this experimentshows that transgenic cES-derived cells persist beyond the embryonicstages and can be seen in juvenile chickens, and that the transgenecontinues to express in these diverse cell types.

The transgene is also present in lymphocytes derived from ES cells. Thehemopeotic lineage, which comprises the lymphoid and myeloid lineages,is of particular interest in transgenic chimeras derived from ES cellsbecause B cells in the lymphoid lineage produce antibodies. Lymphocytesare prepared either from blood samples taken from chimeric chickens atany time from hatching to adulthood or from the bursa of Fabricius ofchimeric embryos. Bursae are removed from chicks at day 20 of embryonicdevelopment (E20) and macerated by forcing through steel mesh in 10 mlof Hanks' Buffered Salt Solution (HBSS) with the plunger of a 20 mlsyringe. The resulting tissue fragments and cells are collected into atube and incubated at room temperature for 5 minutes to allow the largefragments to settle. The cell supernatant is harvested and the cells arecounted and then collected by centrifugation at 1500 g for 10 minutes at4° C. and resuspended at a maximum of 1×10⁸ cells per ml of HBSS in a 15ml conical tube. Blood is collected (0.5 ml) from chimeric chickens'wing veins using a heparinized syringe and deposited into a vacuum tubecontaining EDTA to prevent clotting. Blood samples are mixed 1:1 withHBSS to give a final volume of 1 ml in a 15 ml conical tube. From thispoint on blood samples and bursa samples are treated the same way. Oneml of cell suspension is underlayed with 0.75 ml Fico/Lite-LM (AtlantaBiologicals catalog number 1406) by dispensing the Fico/Lite at thebottom of the tube, underneath the cell suspension. The tubes are thencentrifuged at 1500 g for 15 minutes at 4° C., no brake. The interfacebetween the Fico/Lite and the HBSS is carefully harvested to collect themononuclear cells that have concentrated there in a discrete layer ofmaterial. This material is transferred to a new tube triturated to breakup the compacted cells and then mixed with 3 ml of HBSS/2% heatinactivated fetal bovine serum. The cells are collected bycentrifugation at 1500 rpm in a Sorvall benchtop centrifuge, 10 minutesat 4° C., then washed two more times in HBSS/2% FBS. A small aliquot (25μl) is mounted on a microscope slide for preliminary assessment of theextent of donor-derived GFP fluorescence under the microscope. Theremainder of the cells are then ready for antibody staining or fixation.

To store cells for longer than a few hours before analysis or if theyare to be permanently mounted on slides for microscopy, they are firstfixed in paraformaldehyde. An aliquot of cells (50 μl 0.5-1×10⁶ cells)is fixed by adding 1 ml 4% paraformaldehyde and incubating at roomtemperature for 15 minutes. The cells are then washed three times bycentrifugation at 500 g for 6 minutes in a microfuge to collect thecells, followed by resuspending the cells in PBS/2% heat inactivatedfetal bovine serum.

In antibody staining, an aliquot of 0.5×10⁶ cells in fresh PBS/2%FBS/0.1% sodium azide is placed in a tube or a well of a 96-well plate,on ice. Monoclonal antibodies conjugated to R-phycoerythrin (SouthernBiotechnology Associates) are added to the cells and incubated for 30minutes on ice, covered to protect the fluorophore. Antibodies thatrecognize the chicken B lymphocyte marker Bu-1 (used at a workingdilution of 0.2 g/10⁶ cells) or the chicken T cell marker CD3 (used at0.5 g/10⁶ cells) are used. After incubation the cells are washed threetimes by centrifugation at 500 g for 6 minutes, resuspending in 0.5 mlPBS/2% FBS/0.1% azide each time. After the final wash the cells arestored in 0.5% paraformaldehyde up to one week before analysis by flowcytometry. The paraformaldehyde is replaced with PBS/2% FBS on the daythe cells are to be analyzed by flow cytometry; buffers without phenolred are used for flow cytometry. FACS analysis is performed for both GFPfluorescence and R-phycoerytrin fluorescence simultaneously, to detectthe total proportion of cells that are donor derived (GFP-positive), theproportion of cells (both donor and recipient derived) that are stainedby the antibodies (R-phycoerythrin-positive), and the proportion ofdonor derived cells that are also stained with the antibodies (GFP,R-phycoerytbrin double positive).

Chimeras made with three different ES cell lines were analyzed for donorcontribution to the lymphoid lineage, representing three differentinsertion sites for the CX-GFP marker (Table 9). A total of 27 chimerashave been analyzed, ranging in age from pre-hatch to adult. Theproportion of donor-derived GFP-positive cells in the lymphocytefraction ranged from 0 to 10% as judged by FACS analysis (animals with0% are not shown in the table). Antibody staining of peripheral bloodlymphocytes resulted in 5-17% of the cells staining with anti-Bu-1antibodies and 75-85% of the cells staining with anti-CD3 antibodies.Staining of bursal lymphocytes with anti-Bu-1 antibodies resulted inover 90% of the cells stained. Double positive cells with GFPfluorescence and staining for either Bu-1 or CD3 and GFP were observedat low frequencies in several of the samples.

These data show that ES cells containing a gene encoding an exogenousprotein contributed to the hemopoietic lineage in hatched chicks andmature animals. TABLE 9 Contribution of cES cells to the lymphocytelineage in somatic chimeras. GFP+ and GFP+ and Green Chimera Age GFPBu-1+ CD3+ Bu-1+ CD3+ Sex Feather score Cell line OV-36 10 10 ND ND NDND F 15 ND OVF OV-21 10 7 5 85 0 0 M 0 ND OVF 10881 67 5 17 75 0.03 1 M60 3 TB01 10891 49 3 12 74 0 0.1 F 5 1 TB01 10821 111 1 ND ND ND ND M 753 TB01 10877 98 1 11 ND 0.04 ND F 75 4 TB01 IG1-25 E20 0.2 95 ND 0.03 NDF 70 ND BAC A 10845 146 0.18 8.5 ND 0 ND M 60 3 TB01

The eight chimeras listed in the table were found to contain cEScell-derived lymphocytes. Peripheral blood lymphocytes (or bursallymphocytes in the case of chimera IG1-25) were prepared withFico/Lite-LM as described in the text. Lymphocytes were then analyzedfor expression of green fluorescent protein (GFP), the Bu-1 B cellassociated alloantigen (Bu-1), and the CD3 member of the T cellreceptor-associated CD3 complex (CD3). The numbers in the columnslabeled with GFP, Bu-1 and CD3 indicate the percentage of cells in eachsample that were positive for those markers by FACS analysis (except forOV-36 and 10821 in which the percentage of GFP fluorescing cells wasdetermined by counting cells under the microscope). The age of thechimeras when samples were taken for analysis is indicated in days, andthe sex of each chimera is indicated. The column labeled “Feather”indicates the percentage estimate of black, cES cell derived featherpigmentation, with 75 being the highest amount of black possible (aspure Barred Rock chickens themselves have a plumage that isapproximately 75% black). “Green score” is a subjective evaluation ofoverall green fluorescence in the whole animal visualized by shining ahandheld UV lamp on the live animal. The scale is from 0 (no green) to 4(the most green). This score is used as a further indication of theoverall extent of cES cell contribution in each animal. “Cell line”indicates the names of the different cES cell lines used to generate thechimeras.

There will be various modifications, improvements, and applications ofthe disclosed invention that will be apparent to those of skill in theart, and the present application encompasses such embodiments to theextent allowed by law. Although the present invention has been describedin the context of certain preferred embodiments, the full scope of theinvention is not so limited, but is in accord with the scope of thefollowing claims. All references, patents, or other publications arespecifically incorporated by reference herein.

1. A chicken selectively expressing exogenous protein in tubular glandcells wherein the protein is encoded by a transgene stably integratedinto a genome of the chicken and wherein the transgene is comprised atleast a portion of a promoter of a gene encoding an egg white proteinthat is operably linked to DNA encoding the exogenous protein.
 2. Thechicken of claim 1 further comprising at least a second portion of apromoter of a gene encoding an egg white protein flanking the 3′ end ofthe DNA encoding the exogenous protein.
 3. The chicken of claim 1wherein the exogenous protein is a monoclonal antibody.
 4. The chickenof claim 3 wherein the monoclonal antibody is comprised of a human heavychain.
 5. The chicken of claim 4 wherein the monoclonal antibody isisotype IgG.
 6. The chicken of claim 1 wherein the egg white protein isovalbumin.
 7. The chicken of claim 1 wherein the egg white protein isselected from the group consisting of ovotransferrin, ovomucoid,lysozyme, ovoglobulin G2, ovoglobulin G3, ovoinhibitor, cystatin,ovoglycoprotein, ovoflavoprotein, ovomacroglobulin, and avidin.
 8. Thechicken of claim 1 wherein the size of the transgene is greater than 15kb.
 9. A chicken egg containing a human protein in the egg white,wherein the human protein is encoded by a transgene stable integratedinto the genome of a transgene chicken.
 10. The egg of claim 9 whereinthe human protein is a monoclonal antibody.
 11. A vector comprising apromoter linked to a heterologous coding sequence to express said codingsequence in tubular gland cells of an avian oviduct.
 12. The vector ofclaim 11, wherein said promoter is selected from the group consisting ofan ovomucoid, ovalbumin, lysozyme, and ovomucin promoter.
 13. The vectorof claim 11, further comprising a gene encoding a selectable marker. 14.The vector of claim 11, further comprising an internal ribosome entrysite element.
 15. The vector of claim 11, wherein the heterologouscoding sequence encodes a human antibody.